Cyanobacteria can be modified to produce many types of secondary products, such as biofuels, pharmaceuticals, nutrients, carotenoids, etc. The use of cyanobacteria to produce these products can have several benefits. Cyanobacterial growth does not require the costly input of organic carbon, since cyanobacteria are capable of absorbing light and fixing carbon dioxide as a carbon source for autotrophic growth.
The transformation of the cyanobacterial genus Synechococcus with genes that encode enzymes that can produce ethanol for biofuel production has been described (U.S. Pat. Nos. 6,699,696 and 6,306,639, both to Woods et al.). The transformation of the cyanobacterial genus Synechocystis has been described, for example, in PCT/EP2009/000892, PCT/EP2009/060526, and in U.S. Patent Publication No. US2009/0155871. The cyanobacteria as a whole, however, are a very divergent group of organisms. Due to this diversity, it is often difficult to find a method to effectively and efficiently transform a given host cyanobacterial species. Further, it is also often difficult for the inserted DNA vehicle to replicate adequately once it is present in the host cyanobacterial cell.
Certain strains of cyanobacteria can be naturally transformed. Other cyanobacterial strains can be transformed, for example, by the use of conjugation or electroporation. For a review of cyanobacterial transformation methods, see Vioque, “Transformation of cyanobacteria,” Adv. Exp. Med. Biol. 616:12-22 (2007); Elhai et al., “Conjugal transfer of DNA to cyanobacteria,” Methods in Enzymology 167:747-754 (1988); and Vermaas, “Molecular genetics of the cyanobacterium Synechocystis sp. PCC 6803: Principles and possible biotechnology applications,” Jour. Appl. Phycology 8:263-273 (1996).
One commonly used method of gene transfer to cyanobacteria involves the construction of vectors having a backbone derived from the broad-host range plasmid RSF1010. This plasmid has no cyanobacterial origin of replication, however. The RSF1010-based vector has been widely used as a conjugation vector for transforming bacteria, including cyanobacteria (Mermet-Bouvier et al. (1993) “Transfer and replication of RSF1010-derived plasmids in several cyanobacteria of the genera Synechocystis and Synechococcus” Current Microbiology 27:323-327).
Other vectors for transformation of cyanobacteria include the pDUI-based vectors. The pDU1 origin of replication is best suited for filamentous cyanobacteria, however. Attempts to transform certain species of cyanobacteria with either RSF1010 or pDU1-based shuttle vectors have been unsuccessful.
Several endogenous plasmids from Synechococcus sp. PCC 7002 have been utilized as a backbone plasmid to prepare vectors for heterologous gene expression (Xu et al., Photosynthesis Research Protocols 684:273-293; 2011).
A broad-host-range shuttle vector that replicates in E. coli and three different cyanobacterial strains was developed by Huang et al. Nucleic Acids Research 38:2577-2593 (2010). Expression of three fluorescent reporter proteins (Cerulean, GFPmut3B and EYFP) was demonstrated. Shuttle vectors capable of replication and selection in both E. coli and in the blue green algae Anabaena have been constructed (Wolk et al., PNAS 81:1561-1565 (1984)). Transformation of these vectors apparently requires the presence helper plasmids and a broad host-range plasmid RP-4. These vectors contain regions for replication and mobilization derived from plasmid pBR322, as well as the cyanobacterial replicon pDUI. Other types of vectors for cyanobacteria are described, for example, in Schmetterer et al., Gene, 62:101-109 (1988); Walton et al., Nucleic Acids Research, 21 (3) GenBank Accession No. M81382 (1993); Houmard et al., Methods in Enzymology 167:808-847 (1988).
What is needed in the field of genetically modified cyanobacteria is an easy to manipulate plasmid vector that can be used to express genes of interest in a host cyanobacterial cell, which is capable of being transformed efficiently to a broad range of cyanobacterial species.